Bacterial processes



Patented Aug. 20,

UjNlTEjD- sin-ms PATENT o F-ic BACTERIAL'PROQES SES JacobL. Stokes,Scotch Plains, N. J., assignor to Merck & 00., Inc., Rahway, N. .L, acorporation of New Jersey No Drawings Application October 1, 1942, erialNo. 460,423

7 Claims. (01. 195-96) This invention relates to an improved process forthe production of tyrothricin.

Tyrothricin is a therapeutically important antibacterial substance whichexerts a bactericidal eflect on both gram-positive,and gram-negativemicro-organisms. Tyrothricin is active in extremely small amountsagainst pathogenic bacteria such as Staphylococci; streptococci, andPneumococci. It has also been found effective in the treatment oflocalized infections, and in chronic bovine mastitis.

A method for producing tyrothricin by cultiva tion of an aerobicsporulating bacillus in hydrolized casein, tryptone, or similar media,has been described by Dubos and Dubos and Cataneo (Jr. Exp. 'Med., vol.70, pp. 1-10 and 249-256, 1939). The bacillus used in the describedprocess of the deep cultures, for the production of tyrothricin.

prior art referred to was later identified as Bacillus brevis (Dubos,Jr.'Exp.'Med., vol. 73, pp. 639 640).

That prior art method required the practice of shallow processes, 1. e.,cultivation of the sporulating bacillus in shallow layers (3 cm. deep).Such shallow cultivation processes are disadvantageous in that they aretime-consuming, require an extensive plant, and are less efficient forlargescale commercialpurposes than deep, or sub-. merged processes.

I have now discovered a method whereby tyrothricin may be produced indeep layers, 1. e., submerged cultures. The advantages to be derivedfrom the use of such submerged cultures, as compared to shallow layers,are impressive, and constitute a tremendous economy in space, labor,equipment, and time, with resultant economy in production costs.

'Although hydrolyzed casein, tryptone, yeast extract, and other mediaconsisting primarily of proteins and intermediate products of proteinbreak-down, are suitable for tyrothricin formation in shallow layercultures of Bacillus brevis, tyrothricin cannot be isolated fromdeep-layer or submerged cultures of such media, despite the fact thatexcellent bacterial growth occurs in deep cultures thereof. It appearsthat in deep layers of such media, the metabolism of the bacteria is soaltered that, either tyrothricin is not formed, or possibly, it isformed, but is immediately utilized by the bacteria and does notaccumulate in the medium. Whatever the reason, it is a fact thatnumerous attempts have'been made by me over a prolonged period of timeto isolate tyrothricin from submerged cultures of Bacillus brevis, intryptone medium, for example, without success,

The bacilli are of the"activef group isolated from soil, and composed ofstrains whose broth cultures possess readily demonstrable bactericidalproperties. Such strains are described in Jr. Bacteriology. vol. 43, No.2, February, 1942, pp. 253-' 63; they form H28 in peptone media, do nothydrolyze starch, and are gram-negative in 18- to 24-hour brothcultures.

The synthetic media according to my inven tion comprise mixtures ofinorganic salts, to

which are added a suitable source of carbohydrate, such as glucose, forexample, and a source of nitrogen. usual salts used in synthetic mediafor bacteria processes. However, the source of nitrogen used in thesynthetic media for the purposes oi my invention must be carefullyselected. Not all substances known to be sources of nitrogen aresuitable'for addition to a synthetic medium for the production oftyrothricin in deep cultures. I have found that excellent results areobtained by the submerged process of my invention, it the source ofnitrogen usedin the syntheticmedia is an amino acid selected fromthe'group consist-' ing of B-alanine, dl-phenyl alanine, dl-asparticacid, glycine, dl-glutarnic acid, dl-leucine, laevoproline,laevo-histidine monohydrochloride, and dl-isoleucine, or combinations ofsuch amino acids, as for example, combinations or mixtures comprisingfour of the mentioned amino acids. Ammonium phosphate may also beutilized as a source of nitrogen in a synthetic medium according to myinvention, for the production of tyrothricin in submerged cultures,but'is not as satisfactory as the amino acids specifically mentionedherein. The yield of tyrothricin obtained by cultivating the selectedaerobic sporulating bacillus in a medium containing ammonium phosphateas a source of nitrogen is lower than the yield obtained when thebacillus is cultivated in a synthetic medium containing the designatedamino acids as a source of nitrogen.

The inorganic salts may be the such purposes. I prepared. containingsuch a'mixture comprising fore, may be readily duplicated.

Although amino acids selected from the group consisting of p-alanine,dl-phenyl alanine, dl-as-;

partic acid, glycine, dl-glutamic acid, dl-leucine,

laevo-proline, laevo-histidine mono-hydrochloride, and dl-isoleucine,either individually, or in small groups, may be utilized in a syntheticme- 3 dium according to my invention, for the production of tyrothricinindeep layers, with excellent results, I have found that it isnot-desirable to attempt to utilize a mixture comprising all of the 3mentioned amino acids plus l-cystine, dl-lysine,

l-tryptophane, dl-methionine and l-tryrosine for When a synthetic mediumwas all of the mentioned amino acids, and the medium was inoculated withBacillus brevis or closely related strains of bacilli, as disclosedherein, ty-

rothricin was not obtained from the deep cultures. Furthermore, whensuch a mixture comprising 1 all of the amino acids mentioned above isadded to a synthetic medium which is otherwise satisfactory for thepurposes of my invention, such as a medium comprising a mixture ofinorganic salts, glucose, and dl-glutamic acid, and the medium isinoculated with Bacillus brevis or the like, the formation oftyrothricin is inhibited.

Nor are all amino acids suitable sources of nitrogen for use in asynthetic medium for the production of'tyrothricin in submergedcultures.

Thus, for example, laevo-tyrosine isnot a satisfactory source ofnitrogen for such purposes.

The utilization of the nitrogen source by Bacillus 5 brevis or relatedstrains of bacilli, for the production of tyrothricin in deep cultures,appears to be selective, and many' of the substances known to be sourcesof nitrogen are not suitable for use in j the'process of .my invention.Thus, it appears that Bacillus bre'vis cannot utilize nitrate nitrogen.If urea, ammonium chloride, ammonium nitrate, or mono-ammonium phosphateare sub- 1 stituted in a. synthetic medium for the amino acids disclosedherein, the formation of tyrothricin in deep layers does not occur.

As an example, a synthetic medium according I to my invention maycomprise the following constituents:' calcium mono-phosphate (preferablyin the form of a saturated aqueous solution), potassium hydrophosphate,dihydro-potassium phos-. l phate, magnesium sulfate, sodium chloride,ferrous sulfate, and manganese sulfate; glucose, and 1 dl-glutamic acid.1 s

It will be understood, of course, that modifications may be made in thespecific inorganicsalt Such synthetic media are highly satisfactory fordeep-layer or submerged cultivation of the 1 bacilli, with consequentformation of high yields of tyrothricin. The syntheticmedia of myinvention also have the further advantage that they may be chemicallycharacterized, and there- In carrying out the submerged process of myinvention, the selected synthetic culture medium,

I in sterile condition, is inoculated with a suitable broth culture ofthe selected sporulating aerobic bacillus. It is desirable that theculture be aerated and agitated during the incubation period. Aerationmay be achieved. by passing a stream of air 1 into the culture, and,depending upon the type of chamber in which the process is conducted,agitation may be effected in any appropriate manner, as by stirring,rotating, the use of propeller blades, shaking, etc. The temperature ofthe liter-volume of culture.

4v culture during incubation tained at about 37 C.

The yield of tyrothricin obtained by the submerged process, according tomy invention, compares very favorably with that obtained by theshallow-layer processes of the art, and, in fact,

has been found to be as high as 29% greater per The time required fortyrothricin formation in good yields by my-submerged process is onlyabout one-half the time required in the shallow-layer process. Theproduct obtained by my process exhibits the same in vitro and in vivoactivity as that. produced in shallow cultures in tryptone medium, and,furthermore, generally speaking, is a much finer and whiter product.

The following examples illustrate methods of carrying out the presentinvention, but it is to be understood that these examples are given byway of illustration, and not of limitation.

is adjusted to 7 and is sterilized in a one-liter Erlenmeyer flask. 10guns. of sterilized glucose are added, and the whole inoculated withabout i 0.2% of a tryptone broth cultureof Bacillus brevis.

An aeration tube is inserted into the culture. I

which is agitated and aerated by passing in a slow stream of air,whilebeing incubated at 37 C.

Incubation is allowed to proceed for from 36-60 hours, after which timeprofuse bacterial growth is evident. The pH of the culture is thenadjusted. I to about 4.6-4.8, and a precipitate forms whichconsists ofvegetative cells, spores, cellular debris, and tyrothricin. Theprecipitate is collected by appropriate means, as by centrifugation orfiltration, and suspended in 95% ethyl alcohol (20 cc.

. of alcohol for each liter of culture) for from 18-24 Imixture'illustrated herein, both with respect to the particularinorganic salts, and also with respect to their quantitativerelationships.

hours. The alcoholic-suspension is filtered, and

the alcohol extract evaporated to dryness, ex-

tracted with ether to remove 'fatty impurities;

and redissolved in 95% ethyl alcohol. The alcohol solution ismixed with10 volumes of a 1% sodium chloride solution which. precipitatestyrothricin. The precipitated tyrothricin is collected and-dried invacuo at room temperature,

or at about 37 C. The product, tyrothricin,

occurs inthe form of a fine, white powder.

Example II Fiveliters of a culture medium as described in Example I areplaced in two-gallon glass-lined or carbon steel fermenters' andsterilized; (glucose sterilized separately and added to thesterlleinorganic salt mixture), and the whole-is inoculated with a 2% tryptonebroth culture of Bacillus breois. Aeration is effected by passing in 1.5liters of air per minute, and agitationv is provided by means ofpropeller blades rotating at 60 R. P. M.

The temperature of the culture'during incubation is maintained at about37 C. After frun 36-60 is preferably main tive, only, and my inventionis to be limited only by the appended claims.

I claim: l

1. Ina process for obtaining-an anti-bacterial substance the step thatincludes cultivation of strains of Bacillus brevis, under aerobicsubmerged conditions, in an aqueous medium. containing as a source ofnitrogen a substance selected from the group consisting of B-alanine,d1-

acid, glycine, dl-a-alanine, laevo-hydroxyproline,

dl-threonine, dl-serine, dl-valine, dl-glutamic acid, dl-leucine,laevo-proiine, laevo-histidine monohydrochloride and dl-isoleucine, andrecovering tyrothricin.

3. The process that comprises cultivating strains of Bacillus brevis,"under aerobic submerged conditions, in an aqueous solution-of asynthetic medium comprising a mixture of inorganic salts, a source ofcarbohydrate, and a source of nitrogen selected from the groupconsisting of fl-alanine. dl-phenyl alanine, dl-aspartic acid. glycine.dlu-alanine, laevo-hydroxyproline,

dl-threonine, dl-serine, dl-valine, dl-glutamic acid, dl-leucine,laevo'-proline, laevo-histidine monohydrochloride, and dl-isoleucine,and recovering tyrothricin.

4. In a process for obtaining an anti-bacterial substance the step thatincludes cultivation of strains of Bacillus brem's, under aerobicsubmerged conditions, in an aqueous medium comprising a mixture ofinorganic salts consisting of calcium monophosphate, potassiumhydrophosphate, dihydropotassium phosphate, magnesium sulfate, sodiumchloride, ferrous sulfate, and manganese sulfate, a source ofcarbohydrate, and a source of nitrogen selected from the groupconsisting of fl-alanine, dl-phenyl alanine, di-a'spartic acid, glycine,dl-a-alanine, laevo-hydroxyproline, dl-threonine, dl-serine, dl-valine,dl-glutamic acid, dl-leucine, laevo proline, laevo-histidinemonohydrochloride and dl-isoleucine..

5. In a process for obtaining an anti-bacterial substance the step thatincludes'cultivation of strains of Bacillus brevis, under aerobicsubmerged conditions, in an aqueous medium comprising a mixture ofinorganic salt consisting of calcium monophosphate, potassiumhydrophosphate, dihydro potassium phosphate, magnesium sulfate, sodiumchloride, ferrous sulfate, and manganese sulfate; glucose, and a sourceof nitrogen consisting of dl-glutamic acid. A

6. In a process for obtaining an anti-bacterial substance the step thatincludes cultivation of strains of Bacillus brevis, under aerobicsubmerged conditions, in an aqueous medium comprising a mixture ofinorganic salts, glucose and a 1'sgiurce of nitrogen consisting ofdl-glutamic ac i '7. In a process for obtaining an anti-bacterialsubstance the step that includes cultivation of strains of Bacillusbrevis, under aerobic submerged conditions, in an aqueous mediumcomprising a mixture of inorganic salts, a source of carbohydrate, and asource of nitrogen consisting of dl-glutamic acid.

JACOB L. STOKES.

